Separation science is a universal technique that benefits most scientists. Chromatographic separations provide a means to determine to what extent a reaction has yielded the desired products, to monitor impurities and dissolution profiles, and to study degradation pathways in drug products. Disadvantageously, chromatographic separations are relatively long and tedious processes with analysis times up to approximately one (1) hour or more. Another problem associated with conventional chromatographic separations is the method development time. Screen multiple columns, materials and instruments to achieve optimal separation is labor intensive.
There are a number of types of chromatography systems and generally one way of classifying the types is by operating pressures required to load and move a gradient through an analytical column. For example, chromatography systems are available in gas and liquid form and also at various pressures, such a low pressure systems, high pressure system and ultra-high pressure systems.
One of the disadvantages of conducing ultra-high chromatographic separation is that the equipment must be carefully selected and arranged such that the individual components can withstand the pressures generated in such an operation. This has been a major obstacle in providing an ultra-high pressure chromatography system that is constructed to withstand ultra-high temperatures of greater than 5,000 psi and upwards to 60,000 psi. More specifically, the equipment that is used in a standard LC system is often times unable to be used in a UHPLC setting since it is unsuitable for such an environment and/or is unable to properly function in this type of environment.
For example, a UHPLC pump that can be operatively connected to the solvent reservoir is generally not commercially available as well as one that is capable of gradient elution is likewise not available. Furthermore, performing injection under ultra-high pressures in a conventional LC system requires that the injector be able to withstand such pressures. Unfortunately, commercial injectors can not handle the ultra-high pressures and thus, they are unable to load the injection plug onto the analytical column under the desired ultra-high pressures. Alternatively, static-split flow injectors that can be used in place suffer from a number of disadvantages also, namely, that these type of injectors are of a manual operation type and they operate such that the injected amount is dependent upon the injection pressure and duration of time that the pressure is applied. As a result, the injection volume is difficult to control and estimate. Another challenge concerns the construction of the columns that are used in an UHPLC setting and the dimensions of the columns have to be tailored to permit efficient loading at the desired pressures.
In addition, high performance liquid chromatography (HPLC) has traditionally been performed in columns packed with 3 or 5 μm particle diameters. The internal diameter of these columns is typically between 2 and 4.6 mm, although smaller column diameters are gaining popularity. High separation efficiency with a concomitant gain in analysis time is achieved by reducing particle size and chromatographic packing materials with diameters in the range of 1 μm are now commercially available. However, the use of such materials imposes a great demand on the column inlet pressures that are required to drive the mobile phase through the chromatographic column and since these pressures are greater than those commonly provided by most commercially available pumping devices, the use of such materials is very difficult in a HPLC application. The problem is augmented by the fact that the optimum linear velocity is also dependent on the inverse of the particle diameter, and therefore, the required pressure to operate at optimum velocities is inversely proportional to the cube of the particle diameter.
In an effort to avoid the use of pumping devices, capillary electrochromatography (CEC) has been used to drive the mobile phase through a packed structure, even with submicron particles. However, CEC has presented several problems, including the column fabrication and its reproducibility, and the demands on the packing material to properly participate in the pumping mechanism via electroosmosis while serving as the stationary phase simultaneously, among others.
It is therefore desirable to provide an alternative LC system configured as a UHPLC system that has the ability to perform separations with very high efficiencies and is constructed in a cost effective manner.